A sa-mpn packaged bacteriophage preparation and a preparation method and application thereof

Abstract

The application belongs to the technical field of microbial preparation, and specifically discloses a kind of SA-MPN encapsulated bacteriophage preparation and its preparation method and application, its preparation method includes the following steps: take bacteriophage cocktail concentrate, add tannic acid solution, FeCl3·6H2O solution, phosphate buffer solution, obtain metal phenolic aldehyde network encapsulated bacteriophage;Subsequently, CaCl2 solution and sodium alginate solution are added to the metal phenolic aldehyde network encapsulated bacteriophage in sequence, to obtain sodium alginate-metal phenolic aldehyde network-bacteriophage mixture microcapsules.The application adopts the above-mentioned SA-MPN encapsulated bacteriophage preparation and its preparation method and application, which can effectively protect bacteriophage from gastric acid, high temperature and ultraviolet stress, realize intestinal targeting and slow release, has high encapsulation efficiency, good stability, antioxidant activity, significant antibacterial effect on salmonella and high biological safety.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, and in particular to an SA-MPN encapsulated phage preparation, its preparation method, and its application. Background Technology

[0002] Salmonella is a major pathogen causing intestinal infections, diarrhea, and enteritis in livestock and poultry. In large-scale buffalo farming, it easily leads to outbreaks, severely impacting farming efficiency and livestock product safety. Long-term reliance on antibiotics not only easily induces multidrug-resistant strains but also disrupts the intestinal microecological balance and causes drug residues. Therefore, phage therapy, with its advantages of strong targeting, low resistance rate, and minimal disruption of normal flora, has become a cutting-edge strategy to replace antibiotics in controlling bacterial intestinal infections in livestock and poultry. Among these, phage cocktail therapy has successfully treated cases of multidrug-resistant bacterial infections in clinical practice, demonstrating significant application potential in the prevention and treatment of drug-resistant bacterial infections.

[0003] In existing technologies, oral administration is the mainstream route for phage therapy of intestinal infections, as it can directly target lesions in the digestive tract, is easy to administer, and is suitable for large-scale livestock farming. However, during in vivo delivery of free phages, the highly acidic environment of the stomach (pH < 3) and digestive enzymes rapidly destroy the phage structure, leading to significant inactivation before reaching the target site in the intestine, making it difficult to maintain effective titers for antibacterial effects. Simultaneously, phages are sensitive to external environmental factors such as temperature and ultraviolet radiation, exhibiting poor stability during storage and application, severely limiting the clinical translation and industrial application of oral phage preparations. Furthermore, microencapsulation technology can be used to improve the gastrointestinal tolerance and stability of phages. However, common carrier materials have poor acid resistance, and the constructed encapsulation structure is easily damaged in highly acidic environments, offering limited protection to the phages and causing premature release and inactivation, thus failing to achieve targeted intestinal delivery. Summary of the Invention

[0004] The purpose of this invention is to provide an SA-MPN encapsulated phage formulation, its preparation method, and its application. This formulation can effectively protect phages from gastric acid, high temperature, and ultraviolet stress, achieving targeted sustained release in the intestine. The formulation has a high encapsulation rate, good stability, and antioxidant activity. It also has a significant antibacterial effect against Salmonella and high biosafety.

[0005] To achieve the above objectives, the present invention provides a method for preparing an SA-MPN encapsulated phage formulation, comprising the following steps: S1. Preparation of MPN-Phage complex: Take the phage cocktail concentrate according to the ratio, add tannic acid (TA) solution, vortex mix for the first time, let stand for the first time, then add FeCl3·6H2O solution, vortex mix again, then add phosphate buffer (PBS), vortex mix, let stand again at 24~37℃, wash, and obtain metallophenolic network encapsulated phage (MPN-Phage). S2. Preparation of SA-MPN-Phage mixture microcapsules: Subsequently, CaCl2 solution and sodium alginate (SA) solution were added sequentially to the metallophenolic network encapsulated phage obtained in S1, and the mixture was vortexed to obtain sodium alginate-metallophenolic network-phage mixture microcapsules (SA-MPN-Phage).

[0006] The coordination bond stability of conventional MPN is regulated by the ambient pH. Under acidic conditions, the protonation of the phenolic hydroxyl groups competitively disrupts the metal-phenolic coordination bonds, leading to network dissociation and premature release, resulting in complete phage inactivation. SA, due to its excellent biocompatibility and pH responsiveness, is suitable for encapsulating orally administered phages. When calcium ions interact with the two G segments on the SA molecular chain through four coordination bonds, they form an "eggbox" structure. This structure transforms the adjacent molecular chains from random clusters into a curved, ribbon-like structure cross-linked by calcium ions, constructing a stable three-dimensional gel network that resists gastric acid attack, allowing the phage to pass through gastric juice. When SA-MPN-Phage enters SIF, the phosphate ions in SIF compete with SA for calcium. 2+ Calcium phosphate precipitate is formed. SA also dissolves rapidly in the pH environment of SIF, thereby releasing MPN-Phage. In the pH environment of SIF, the phenolic hydroxyl groups of TA are deprotonated to a higher degree, reacting with Fe. 3+ The coordination of the TA molecules remains relatively stable, so the MPN does not dissociate as quickly as it does in gastric acid. However, trypsin can enzymatically degrade the TA molecules, thereby directly destroying the network structure of the MPN and releasing the phage.

[0007] Preferably, in S1, the phage cocktail concentrate, tannic acid solution, and FeCl3·6H2O solution are mixed, and then mixed with the phosphate buffer at a volume ratio of 1:1, wherein the potency of the phage cocktail concentrate is 10. 8 ~10 10 The concentration of PFU / mL is 30~50 mg / mL for the tannic acid solution and 5~15 mg / mL for the FeCl3·6H2O solution.

[0008] In step S2, the CaCl2 solution and the sodium alginate solution are added at a volume ratio of 1:1, wherein the mass fraction of the CaCl2 solution is 0.2~0.6% and the mass fraction of the sodium alginate solution is 0.2~0.8%.

[0009] Preferably, the concentration of the tannic acid solution is 40 mg / mL, the concentration of the FeCl3·6H2O solution is 10 mg / mL, the mass fraction of the CaCl2 solution is 0.2%, and the mass fraction of the sodium alginate solution is 0.8%.

[0010] Preferably, in S1, the initial vortex mixing time is 20-40 seconds, and the second vortex mixing time is 1 minute.

[0011] Preferably, in S1, the initial settling temperature is 18~25℃ and the settling time is 10min, and the second settling temperature is 24~37℃ and the settling time is 30min.

[0012] Preferably, in S1, the washing is repeated 2-3 times, and the washing process is specifically as follows: Centrifuge at 8000~12000 r / min for 10 min, discard the supernatant, and resuspend the precipitate in sterile phosphate buffer.

[0013] Preferably, in S2, the vortex mixing time is 1 minute.

[0014] The present invention also provides an SA-MPN encapsulated phage formulation.

[0015] This invention also provides an application of SA-MPN encapsulated phage formulation in livestock and poultry feed.

[0016] The present invention also provides the application of an SA-MPN encapsulated phage formulation in the preparation of drugs for diarrhea and enteritis caused by salmonellosis.

[0017] Therefore, the present invention employs the above-mentioned SA-MPN encapsulated phage preparation, its preparation method, and its application, and the beneficial effects are as follows: The preparation process of this invention is mild and suitable for large-scale preparation. The prepared SA-MPN-Phage microcapsules have high encapsulation efficiency, uniform particle size, and good dispersibility. They do not damage the activity of bacteriophages and can significantly enhance the tolerance of bacteriophages to high temperature and ultraviolet light. They can still maintain the bacteriophage titer at 60~70℃ and do not show significant inactivation after 120 minutes of ultraviolet irradiation, which greatly improves the stability of the formulation in storage and application.

[0018] The SA-MPN-Phage microcapsules prepared in this invention have an outer sodium alginate-calcium ion hydrogel that can resist the erosion of simulated gastric juice, preventing the rapid inactivation of bacteriophages in the highly acidic environment of the stomach. After entering simulated intestinal fluid, the carrier material gradually degrades in response to pH and enzymatic hydrolysis, achieving slow and complete release of bacteriophages and ensuring targeted and sustained-release efficacy in the intestine. They are used to prepare drug formulations for diarrhea and enteritis caused by salmonellosis, and have a significant in vitro inhibitory effect on Salmonella. The hemolysis rate is <3%, and there is no obvious acute toxicity or organ damage in mice. They have excellent biocompatibility and meet the safety requirements for oral formulations.

[0019] The SA-MPN-Phage microcapsules prepared by this invention have good DPPH and ABTS free radical scavenging capabilities, which can synergistically reduce intestinal oxidative stress and can be added to livestock and poultry feed to improve intestinal health.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This is an EDTA release curve diagram of Embodiment 1 of the present invention; Figure 2 This is the Fourier transform infrared spectrum of Embodiment 1 of the present invention; Figure 3 This is a temperature stability result graph of Embodiment 1 of the present invention, where A is the temperature stability result for 30 minutes and B is the temperature stability result for 1 hour. Figure 4 This is the UV stability curve of Embodiment 1 of the present invention; Figure 5 This is a stability curve of simulated gastric juice (SGF) in Embodiment 1 of the present invention; Figure 6 This is the simulated bile salt (SBF) stability curve of Example 1 of the present invention; Figure 7 This is a simulated intestinal fluid (SIF) release curve diagram of Embodiment 1 of the present invention; Figure 8 This is a graph of DPPH free radical scavenging kinetics from Embodiment 1 of the present invention, where A represents free TA and B represents SA-MPN; Figure 9 This is a comparison chart of DPPH free radical scavenging results in Example 1 of the present invention; Figure 10 This is a graph of the ABTS radical scavenging kinetics of Embodiment 1 of the present invention, where A is free TA and B is SA-MPN; Figure 11 This is a comparison chart of ABTS free radical scavenging results in Example 1 of the present invention; Figure 12This is a diagram illustrating the in vitro antibacterial effect of Embodiment 1 of the present invention; Figure 13 This is a graph showing the hemolysis test results of Example 1 of the present invention, where A is a visual graph of hemolysis and B is a statistical graph of hemolysis rate; Figure 14 These are histopathological sections of mouse organs and tissues from Example 1 of the present invention, where A is the heart organ index of the control group, B is the liver organ index of the control group, C is the spleen organ index of the control group, D is the lung organ index of the control group, E is the kidney organ index of the control group, F is the heart organ index of the SA-MPN-Phage group, G is the liver organ index of the SA-MPN-Phage group, H is the spleen organ index of the SA-MPN-Phage group, I is the lung organ index of the SA-MPN-Phage group, and J is the kidney organ index of the SA-MPN-Phage group. Figure 15 This is a comparison chart of mouse organ indices in Example 1 of the present invention, where A is the heart organ index, B is the liver organ index, C is the spleen organ index, D is the lung organ index, and E is the kidney organ index. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0025] The Phage cocktail phage used in all embodiments of this invention was sourced from wastewater from a large-scale water buffalo farm in Tiandong County, Guangxi Zhuang Autonomous Region.

[0026] Example 1 An SA-MPN encapsulated phage formulation is prepared as follows: S1. Preparation of MPN-Phage complex: In a clean bench, take 490µL of a 10-potency... 10 PFU / mL Phagecocktail concentrate was slowly added to 5µL TA solution under vortexing. The mixture was vortexed for 30 seconds initially and allowed to stand at room temperature for 10 minutes. Then, 5µL FeCl3·6H2O solution was added under vortexing and vortexed again for 1 minute. 500µL PBS was then added and gently vortexed to mix. The mixture was allowed to stand at 37°C for 30 minutes and then centrifuged at 10000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was resuspended in sterile PBS. The above washing process was repeated twice to remove unsuccessfully or incompletely encapsulated phages, resulting in MPN-Phage. S2. Preparation of SA-MPN-Phage mixture microcapsules: Subsequently, 100µL CaCl2 solution and 100µL SA solution were added sequentially to the MPN-Phage obtained in S1, and the mixture was vortexed for 1 min to obtain SA-MPN-Phage.

[0027] In this embodiment, the concentration of TA is 40 mg / mL, the concentration of FeCl3·6H2O solution is 10 mg / mL, the mass fraction of CaCl2 solution is 0.2%, and the mass fraction of SA solution is 0.8%.

[0028] Example 2 An SA-MPN encapsulated phage formulation differs from Example 1 in that the concentration of TA is 40 mg / mL, the concentration of FeCl3·6H2O solution is 10 mg / mL, the mass fraction of CaCl2 solution is 0.4%, and the mass fraction of SA solution is 0.5%.

[0029] Example 3 An SA-MPN encapsulated phage formulation differs from Example 1 in that the concentration of TA is 40 mg / mL, the concentration of FeCl3·6H2O solution is 10 mg / mL, the mass fraction of CaCl2 solution is 0.6%, and the mass fraction of SA solution is 0.5%.

[0030] Example 4 An SA-MPN encapsulated phage formulation differs from Example 1 in that the concentration of TA is 40 mg / mL, the concentration of FeCl3·6H2O solution is 5 mg / mL, the mass fraction of CaCl2 solution is 0.4%, and the mass fraction of SA solution is 0.8%.

[0031] Example 5 An SA-MPN encapsulated phage formulation differs from Example 1 in that the concentration of TA is 50 mg / mL, the concentration of FeCl3·6H2O solution is 15 mg / mL, the mass fraction of CaCl2 solution is 0.2%, and the mass fraction of SA solution is 0.8%.

[0032] Comparative Example 1 An MPN is prepared by the following method: In a clean bench, take 490 µL of PBS and slowly add 5 µL of TA solution while vortexing. Vortex for 30 seconds and let stand at room temperature for 10 minutes. Then, add 5 µL of FeCl3·6H2O solution while vortexing and mix for 1 minute. Add 500 µL of PBS, gently vortex to mix, and let stand at 37°C for 30 minutes. Centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, and resuspend the precipitate in sterile PBS to obtain MPN.

[0033] Comparative Example 2 An SA-MPN is prepared as follows: In a clean bench, 490 µL of PBS was added slowly with 5 µL of TA solution under vortexing for 30 seconds, and then allowed to stand at room temperature for 10 minutes. Next, 5 µL of FeCl3·6H2O solution was added under vortexing for 1 minute, followed by the addition of 500 µL of PBS. The mixture was then gently vortexed and allowed to stand at 37°C for 30 minutes. After centrifugation at 10000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile PBS to obtain MPN. Subsequently, 100 µL of CaCl2 solution and 100 µL of SA solution were added sequentially to the MPN, and the mixture was vortexed for 1 minute to obtain SA-MPN.

[0034] Test 1. The encapsulation efficiency, particle size, zeta potential, and polydispersity index (PDI) of microencapsulated phages were used as evaluation indicators. The encapsulation efficiency was calculated using the following formula: .

[0035] A four-factor, three-level orthogonal experimental design was adopted. The orthogonal experimental table is shown in Table 1, and the orthogonal experimental results are shown in Table 2. Range analysis was performed on the orthogonal experimental results. The range analysis tables for Zeta potential, PDI, encapsulation efficiency, and particle size are shown in Table 3, 4, 5, and 6, respectively, to determine the optimal preparation conditions.

[0036] Table 1 Orthogonal Experiment Table

[0037] Table 2. Results of Orthogonal Experiments

[0038] Table 3 Zeta Potential Range Analysis Table

[0039] Table 4 PDI Range Analysis Table Table 5 Encapsulation Rate Range Analysis Table

[0040] Table 6. Particle Size Range Analysis Table

[0041] As shown in Tables 1 to 6, the order of influence of each factor on particle size is: FeCl3·6H2O>TA>CaCl2>SA; Zeta potential: FeCl3·6H2O>TA>SA>CaCl2; PDI: TA>CaCl2>FeCl3·6H2O>SA; Encapsulation efficiency: FeCl3·6H2O>TA>SA>CaCl2. Based on particle size as the primary evaluation criterion, the optimal formulation is TA 2, FeCl3·6H2O 2, SA 1, and CaCl2 1; based on potential as the primary evaluation criterion, the optimal formulation is TA 2, FeCl3·6H2O 2, SA 2, and CaCl2 3; based on PDI as the primary evaluation criterion, the optimal formulation is TA 2, FeCl3·6H2O 1, SA 3, and CaCl2 2; based on encapsulation efficiency as the primary evaluation criterion, the optimal formulation is TA 3, FeCl3·6H2O 3, SA 3, and CaCl2 1. Since the four indicators are not ranked in terms of importance, a comprehensive balance method was ultimately adopted to select the optimal formulation, which is TA 2, FeCl3·6H2O 2, SA 3, and CaCl2 1.

[0042] 2. The release of the Phage cocktail and the MPN-Phage and SA-MPN-Phage prepared in Example 1 were detected in ethylenediaminetetraacetic acid (EDTA) using the following method: Add 100 μL of Phage cocktail, MPN-Phage, and SA-MPN-Phage to centrifuge tubes containing 4.9 mL and 200 mM EDTA solution, respectively. Place the tubes on a constant temperature shaker (37℃, 180 rpm). Vortex the samples for 30 s every 10 min, then pipette 100 μL of the culture medium and centrifuge at 10000 rpm for 10 min. Collect the supernatant to determine the titer. Incubate for 1 h. Results are as follows: Figure 1 As shown.

[0043] Depend on Figure 1 It can be seen that the titer of the Phage cocktail group phages in EDTA did not change significantly. The MPN-Phage group and the SA-MPN-Phage group started to release at 10 min, the MPN-Phage group completed release at 30 min, and the SA-MPN-Phage group completed release at 50 min.

[0044] 3. The SA-MPN-Phage prepared in Example 1 was subjected to Fourier transform infrared spectroscopy. The detection method is as follows: Take 50 mg of the pre-prepared lyophilized SA-MPN-Phage sample and mix it with pure potassium bromide at a ratio of 1:100. Grind the mixture thoroughly in a grinding mortar. Take a small amount of the ground product and press it into a pellet. After checking the transmittance of the sample pellet, place it in a Fourier transform infrared spectrometer and set the scanning range to 400–4000 cm⁻¹. -1 The test results are as follows Figure 2 As shown.

[0045] Depend on Figure 2 It can be inferred that TA is at approximately 3400cm. -1 A typical broad and strong absorption peak appears at 3400 cm⁻¹, which is due to the stretching vibration of the phenolic hydroxyl group (–OH) and intermolecular hydrogen bonding. When TA reacts with FeCl₃·6H₂O to form MPN, a peak appears at 3400 cm⁻¹. -1 The peak at 1600 cm⁻¹ broadens significantly and shifts towards lower wavenumbers, while at 1600 cm⁻¹... -1 and 1200cm -1 The shape and intensity of the nearby peaks also changed significantly, forming a typical metal polyphenol coordination network structure. The characteristic peak of SA is located at approximately 1600 cm⁻¹. -1 and 1410cm -1 At each location, there is a corresponding carboxylate group (–COO). - The asymmetric and symmetric stretching vibrations of the ions exhibit shifts and intensities after crosslinking with CaCl2, forming the characteristic bands of SA-Ca and demonstrating the formation of the ion-crosslinked hydrogel network. In the spectrum of the final SA-CaCl2-MPN product, both the characteristic peaks of MPN and SA-Ca can be observed simultaneously, proving the successful preparation of SA-MPN.

[0046] 4. The temperature stability of the Phage cocktail and the MPN-Phage and SA-MPN-Phage prepared in Example 1 were determined using the following methods: Pre-prepared lyophilized samples were randomly divided into six temperature groups, with six samples in each group. These groups were incubated in water baths at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, respectively, for 30 min and 60 min incubation times. After incubation, the centrifuge tubes were immediately removed and 900 µL of cooled LB liquid medium was added. The phage titer was determined using the bilayer plate method. The experiment was repeated three times, and the results are shown below. Figure 3 As shown, "ns" indicates no significant difference ( P >0.05); "*" indicates a significant difference ( P<0.05); "**" indicates a highly significant difference ( P <0.01).

[0047] Depend on Figure 3 It can be seen that when incubated at 30-50℃ for 30 minutes to 1 hour, the titers of all three phage groups remained stable. Figure 3 As shown in A, after treatment at 60-80℃ for 30 min, the MPN-Phage group showed no significant difference in potency compared to the Phage cocktail group, while the SA-MPN-Phage group showed a highly significant increase in potency. P <0.01); and the titer of the SA-MPN-Phage group was significantly higher than that of the MPN-Phage group ( P <0.01). After incubation at 80℃ for 30 min, the phages in the Phage cocktail group and the MPN-Phage group were completely inactivated, while approximately 54 PFU / mL could still be detected in the SA-MPN-Phage group.

[0048] like Figure 3 As shown in B, extending the incubation time to 1 hour and incubating at 60℃ resulted in a significantly higher titer in the SA-MPN-Phage group compared to the other two groups. P <0.01). After incubation at 70℃ for 1 hour, the phages in the Phage cocktail group and the MPN-Phage group were completely inactivated, while the titer of the SA-MPN-Phage group remained at approximately 3.1 × 10⁻⁶. 2 PFU / mL. When the temperature was raised to 80℃, all grouped phages were completely inactivated.

[0049] 5. The UV stability of the Phage cocktail and the MPN-Phage and SA-MPN-Phage prepared in Example 1 was determined using the following method: Add the lyophilized sample to a 60mm diameter petri dish, open the lid, and place it 40cm away from a 30W UV lamp for 120 minutes of continuous irradiation. Take 100µL of phage enrichment solution every 20 minutes and determine the phage titer using the bilayer plate method. Repeat the experiment three times. The results are as follows: Figure 4 As shown.

[0050] Depend on Figure 4 It can be seen that the initial titer of the three groups of bacteriophages was approximately 4.1 × 10⁻⁶. 10At 60 min, the phage titer decreased by one order of magnitude at PFU / mL, and by two orders of magnitude at 100 min. The phage titer in the Phage cocktail group decreased slowly over time. The phage titers in the MPN-Phage and SA-MPN-Phage groups showed a decreasing trend within 120 min, but without a significant decrease.

[0051] 6. In vitro simulation tests were performed on the Phage cocktail and the MPN-Phage and SA-MPN-Phage prepared in Example 1, including the stability of simulated gastric fluid (SGF), the stability of simulated bile fluid (SBF), and the release of simulated intestinal fluid (SIF).

[0052] a. The method for determining the stability of SGF is as follows: 1 mL of Phage cocktail, MPN-Phage, and SA-MPN-Phage were added to 4 mL of simulated gastric fluid (pH=2) preheated to 37°C, and incubated on a 37°C shaker at 180 rpm for 2 h. 200 μL of co-culture were collected at 0, 5, 10, 15, 30, 60, 90, and 120 min, respectively, and phage titers were determined using the bilayer plate method. The experiment was repeated three times, and the results are shown below. Figure 5 As shown.

[0053] Depend on Figure 5 It was found that in the Phage cocktail group and the MPN-Phage group, the phage titer decreased by about three orders of magnitude at 5 minutes, and the phage was completely inactivated at 15 minutes. In the SA-MPN-Phage group, the phage titer was relatively stable in the first 5 minutes, began to decrease at 10 minutes, and decreased rapidly at 15 minutes, decreasing by about three orders of magnitude compared with the initial titer, and the phage was inactivated at 90 minutes.

[0054] b. The method for determining the stability of SBF is as follows: Add 1 mL of Phage cocktail, MPN-Phage, and SA-MPN-Phage to 4 mL of simulated bile salts preheated to 37°C, respectively, and incubate at 180 rpm for 2 h in a 37°C constant-temperature shaker. Take 200 μL of co-culture every 30 min and determine the phage titer using the double-layer plate method. Repeat the experiment three times. The results are as follows: Figure 6 As shown.

[0055] Depend on Figure 6It can be seen that the titer of the bacteriophages in the Phage cocktail group, MPN-Phage group and SA-MPN-Phage group was not significantly reduced under the action of simulated bile salts.

[0056] c. The method for determining the stability of SIF is as follows: 1 mL of Phage cocktail, MPN-Phage, and SA-MPN-Phage were added to 4 mL of simulated intestinal fluid preheated to 37°C, respectively, and incubated on a 37°C shaker at 180 rpm for 7 h. Samples were taken every 20 min for the first 3 h, and every 1 h for the next 4 h. The samples were centrifuged at 10000 rpm for 10 min, and the supernatant was collected. Phage titers were determined using the double-layer plate method. The experiment was repeated three times, and the results are as follows: Figure 7 As shown.

[0057] Depend on Figure 7 It can be seen that the phage titer in the Phage cocktail group remained stable in SIF, while both the MPN-Phage group and the SA-MPN-Pzhage group began to release phages at 20 min, with titers of approximately 1.73 × 10⁻⁶. 6 PFU / mL and 5.1×10 4 PFU / mL. The MPN-Phage group was completely released at 300 min, while the SA-MPN-Phage group was completely released at 360 min, indicating that the release rate of the MPN-Phage group was higher than that of the SA-MPN-Phage group.

[0058] 7. The free radical scavenging properties of the MPN-Phage and SA-MPN-Phage prepared in Example 1 were determined, including DPPH free radical scavenging detection and ABTS free radical scavenging detection. Freshly prepared MPN-Phage and SA-MPN-Phage were taken and tested according to the instructions of the Total Antioxidant Capacity Assay Kit - Enzyme-linked Immunosorbent Assay (DPPH Method). The results are as follows: Figure 8 and 9 As shown, the test was performed according to the instructions of the Total Antioxidant Capacity Assay Kit - Enzyme-Based Immunosorbent Assay (ABTS method), and the results are as follows. Figure 10 and 11 As shown, "ns" indicates no significant difference ( P >0.05), "*" indicates a significant difference ( P <0.05), "**" indicates a highly significant difference ( P <0.01).

[0059] Depend on Figure 8 As shown in A, the DPPH free radical scavenging rate of free TA reached its peak at 10 min for all concentration groups; Figure 8As shown in B, SA-MPN achieved its peak DPPH free radical scavenging rate for all concentration groups at 5 minutes. Figure 9 It can be seen that the DPPH free radical scavenging rate is directly proportional to the concentration of the formulation material, and its color changes from purple to yellow as the scavenging rate increases. At formulation material concentrations of 600 μg / mL and 400 μg / mL, the DPPH scavenging rate of the SA-MPN group was significantly higher than that of the free TA group (…). P <0.05); no statistically significant difference was found in the other concentration groups ( P >0.05), but the clearance rate of the SA-MPN group was still generally higher than that of the free TA group.

[0060] Depend on Figure 10 As shown in A, the ABTS radical scavenging rate of the free TA group reached its peak at 5 minutes. Figure 10 As shown in B, in the SA-MPN group, the ABTS free radical scavenging rate reached its peak at 5 minutes. Figure 11 It can be seen that the ABTS free radical scavenging rate is directly proportional to the concentration of the formulation material, and its color changes from blue to white as the scavenging rate increases. At a formulation material concentration of 100 μg / mL, the DPPH free radical scavenging rate of the SA-MPN group was significantly higher than that of the free TA group (…). P <0.01); no statistically significant difference was found in the other concentration groups ( P >0.05), but the clearance rate of the SA-MPN group was still generally higher than that of the free TA group.

[0061] 8. In vitro antibacterial activity assays were performed on Phage cocktail, MPN, SA-MPN, and MPN-Phage and SA-MPN-Phage prepared in Example 1. The assay method is as follows: Add 7 mL of LB liquid culture medium to a 10 mL centrifuge tube to set up a positive control group ( Salmonella The study included a positive control group, a negative control group, and a sodium alginate-metallophenolic network group. The positive control group received 700 µL of logarithmically growing Salmonella GXU 2603 bacterial suspension; the negative control group received 700 µL of PBS solution; and the sodium alginate-metallophenolic network group received 700 µL of logarithmically growing Salmonella GXU 2603 bacterial suspension along with lyophilized samples. Samples were incubated at 37°C and 180 rpm in a shaker. For the first 6 hours, 100 µL of co-culture medium was collected every 30 minutes, and the optical density was measured at 600 nm using a 96-well plate. Measurements were then taken every 12 hours thereafter, and the incubation period was continued for 48 hours. The experiment was repeated three times. Results are shown below. Figure 12 As shown.

[0062] Depend on Figure 12It can be seen that Salmonella GXU 2603 exhibits a logarithmic growth trend. The OD values ​​of the SA-MPN and MPN groups within 1.5 hours were... 600 Remain stable, OD at 1.5h 600 The growth trend was slow. The OD values ​​of the MPN-Phage group, Phage cocktail group, and SA-MPN-Phage group were [data missing] within 2 hours. 600 Remain stable, OD at 2.5h 600 The values ​​showed a slow increasing trend. After 48 hours of continuous monitoring, the OD600 values ​​of the SA-MPN-Phage group, MPN-Phage group, Phage cocktail group, SA-MPN group, and MPN group remained consistently below [value missing]. Salmonella Group.

[0063] 9. Hemolytic activity was determined for PBS (-), Triton X-100 (+), MPN (1), SA-MPN (3), and MPN-Phage (2) and SA-MPN-Phage (4) prepared in Example 1. The determination method is as follows: Blood was collected from the fundus venous plexus of mice. Red blood cells were separated by centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed three times with sterile physiological saline. The washed red blood cells were diluted to 2% (w / t) for later use. Appropriate amounts of MPN, MPN-Phage, SA-MPN, and SA-MPN-Phage were placed in the red blood cell suspension and incubated at 37°C for 2 h. After the reaction was complete, the cells were centrifuged at 5000 rpm for 5 min, and the supernatant was collected for later use. The red blood cells were then analyzed using an ELISA reader at OD500. 540 The absorbance of the supernatant from different groups was measured. Red blood cells incubated with PBS served as a negative control, while those incubated with Triton X-100 served as a positive control. Results are as follows: Figure 13 As shown. The formula for calculating the hemolysis rate is as follows: Depend on Figure 13 As shown in A, the supernatants of the MPN, MPN-Phage, SA-MPN, SA-MPN-Phage, and PBS groups were all colorless and transparent, with almost no erythrocyte lysis. The erythrocytes in the Triton X-100 group were completely lysed and appeared red. Figure 13 As can be seen from B in the figure, based on the detected OD 540 The hemolysis rate of each group was calculated. The hemolysis rate of the Triton X-100 group was approximately 100%, while the hemolysis rate of the other groups did not exceed 3%.

[0064] 10. The in vivo safety of the SA-MPN-Phage prepared in Example 1 was determined in mice. The determination method is as follows: Twelve 6-week-old Kunming mice were selected. Before the experiment, the mice were placed in the animal room and fed normally for 7 days with free access to water and food to minimize stress. Six Kunming mice were selected and administered 200 μL of SA-MPN-Phage orally by gavage daily for 7 consecutive days. The control group received an equal volume of PBS orally. After 7 days, the mice were weighed, their abdomens were disinfected with 75% alcohol, and they were dissected on ice. The ratio of internal organ mass to body weight was calculated for each group, and statistical analysis was performed. The formula for calculating the organ index is as follows: After fixing appropriately sized organ tissues in 4% paraformaldehyde solution for 24 hours, paraffin sections were prepared and stained with hematoxylin and eosin (HE). Figure 14 As shown, the organ structures of the experimental mice were intact and clearly defined, with no obvious inflammatory infiltration or necrosis. Figure 15 As shown, the organ indices of the experimental mice were not significantly different from those of healthy mice. P >0.05).

[0065] Therefore, the present invention employs the above-mentioned SA-MPN encapsulated phage formulation and its preparation method and application, which can effectively protect the phage from gastric acid, high temperature and ultraviolet stress, and achieve targeted sustained release in the intestine; the formulation has high encapsulation rate, good stability, and antioxidant activity, with significant antibacterial effect against Salmonella, and high biosafety.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an SA-MPN encapsulated phage formulation, characterized in that, Includes the following steps: S1. Preparation of MPN-Phage complex: Take the phage cocktail concentrate according to the ratio, add tannic acid solution, vortex mix for the first time, let stand for the first time, then add FeCl3·6H2O solution, vortex mix again, then add phosphate buffer, vortex mix, let stand again at 24~37℃, wash, and obtain metal phenolic network encapsulated phage. S2. Preparation of SA-MPN-Phage mixture microcapsules: Subsequently, CaCl2 solution and sodium alginate solution were added sequentially to the metal phenolic network encapsulated phage obtained in S1, and the mixture was vortexed to obtain sodium alginate-metal phenolic network-phage mixture microcapsules.

2. The method for preparing an SA-MPN encapsulated phage formulation according to claim 1, characterized in that, In S1, the phage cocktail concentrate, tannic acid solution, and FeCl3·6H2O solution are mixed, and then mixed with the phosphate buffer at a volume ratio of 1:

1. The potency of the phage cocktail concentrate is 10. 8 ~10 10 The concentration of the tannic acid solution is 30-50 mg / mL, and the concentration of the FeCl3·6H2O solution is 5-15 mg / mL. In S2, the CaCl2 solution and the sodium alginate solution are added at a volume ratio of 1:1, wherein the mass fraction of the CaCl2 solution is 0.2~0.6% and the mass fraction of the sodium alginate solution is 0.2~0.8%.

3. The method for preparing an SA-MPN encapsulated phage formulation according to claim 2, characterized in that, The concentration of the tannic acid solution is 40 mg / mL, the concentration of the FeCl3·6H2O solution is 10 mg / mL, the mass fraction of the CaCl2 solution is 0.2%, and the mass fraction of the sodium alginate solution is 0.8%.

4. The method for preparing an SA-MPN encapsulated phage formulation according to claim 1, characterized in that, In S1, the initial vortex mixing time is 20~40s, and the second vortex mixing time is 1min.

5. The method for preparing an SA-MPN encapsulated phage formulation according to claim 1, characterized in that, In S1, the initial settling temperature is 18~25℃ and the time is 10min, and the second settling temperature is 24~37℃ and the time is 30min.

6. The method for preparing an SA-MPN encapsulated phage formulation according to claim 1, characterized in that, In S1, the washing is repeated 2-3 times, and the washing process is as follows: Centrifuge at 8000~12000 r / min for 10 min, discard the supernatant, and resuspend the precipitate in sterile phosphate buffer.

7. The method for preparing an SA-MPN encapsulated phage formulation according to claim 1, characterized in that, In S2, the vortex mixing time is 1 minute.

8. An SA-MPN-encapsulated phage preparation prepared by the method of any one of claims 1 to 7.

9. The application of the SA-MPN encapsulated phage formulation as described in claim 8 in livestock and poultry feed.

10. The use of the SA-MPN encapsulated phage formulation as described in claim 8 in the preparation of a drug for diarrhea and enteritis caused by salmonellosis.

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